Project Summary The gold-standard for measuring total daily energy expenditure (TDEE) in free-living individuals is the doubly labeled water (DLW) method, which is based on the principle that differential elimination rates of hydrogen and oxygen following ingestion of a labeled dose of heavy water ( H2 O) is a measure of carbon dioxide (CO2) 2 18 production. However, widespread adoption of the DLW method has been limited by costs (both 18O dose and isotopic analysis costs) and challenges related to sample collection, preparation, and analysis using isotope ratio mass spectrometry (IRMS). Recent technological advances have led to the development of alternative approaches to measuring isotopic enrichments in human samples including off-axis integrated cavity output spectroscopy (OA-ICOS), which combines the use of narrowband continuous wave lasers with optical cavities to measure isotopic enrichments based on absorption spectroscopy. We have shown that OA-ICOS is accurate and precise compared to the gold-standard of whole-room indirect calorimetry, as well as against an independent IRMS laboratory. In this research, we aim to demonstrate that combining simple collection and preparation procedures of saliva and breath with isotopic measurements performed using OA-ICOS provides a valid, more rapid, and less expensive approach compared to IRMS. The natural abundances of 2H:1H and 18O:16O of simultaneously collected urine, saliva, and breath samples will be determined from 50 relatively healthy human subjects. Saliva will be prepared using a patented rapid sample preparation technique, zinc sulfate precipitation of proteins followed by centrifugation. Urine will be prepared using centrifugation only, as previously demonstrated. Water isotopes in breath will be directly measured from collected exhaled breath and adjusted using measured fractionation factors. We will also measure 2H:1H and 18O:16O enrichments in urine, saliva, and breath of 10 relatively healthy human subjects after consuming a standard DLW dose and compare the estimates of TDEE using these different matrices. Validation of the proposed sample collection and analysis procedures will make it possible or more labs to perform DLW measurements. Moreover, because the collection of saliva and breath samples is easier and faster than obtaining urine or serum blood samples, participant burden will be reduced. If successful, we will demonstrate that combing simple collection and preparation procedures of saliva and breath with isotopic measurements performed using OA-ICOS will provide a valid, more rapid, and less expensive approach (<$20/sample) compared to standard IRMS analysis (~$75/sample).